A single ubiquitin is sufficient for cargo protein entry into MVBs in the absence of ESCRT ubiquitination

Abstract

ESCRTs (endosomal sorting complexes required for transport) bind and sequester ubiquitinated membrane proteins and usher them into multivesicular bodies (MVBs). As Ubiquitin (Ub)-binding proteins, ESCRTs themselves become ubiquitinated. However, it is unclear whether this regulates a critical aspect of their function or is a nonspecific consequence of their association with the Ub system. We investigated whether ubiquitination of the ESCRTs was required for their ability to sort cargo into the MVB lumen. Although we found that Rsp5 was the main Ub ligase responsible for ubiquitination of ESCRT-0, elimination of Rsp5 or elimination of the ubiquitinatable lysines within ESCRT-0 did not affect MVB sorting. Moreover, by fusing the catalytic domain of deubiquitinating peptidases onto ESCRTs, we could block ESCRT ubiquitination and the sorting of proteins that undergo Rsp5-dependent ubiquitination. Yet, proteins fused to a single Ub moiety were efficiently delivered to the MVB lumen, which strongly indicates that a single Ub is sufficient in sorting MVBs in the absence of ESCRT ubiquitination.

Figure 1.

Vps27 lacking lysine residues is not ubiquitinated but still functions in MVB cargo sorting. (A) Ubiquitination of Vps27 was assessed in cells expressing HA-Vps27 and myc-Ub. Vps27 immunoprecipitates (IP) from denatured cell lysates were immunoblotted (IB) for HA-Vps27 (anti-HA) or Ub (anti-myc). Input represents a 5% equivalent. Black lines indicate that dividing lanes have been spliced out. (B) Domain organization of yeast ESCRT-0 protein Vps27. Blue lines show positions of the 47 lysines. (C) Ubiquitination of wild-type and lysine-less Vps27 (HA-Vps27^WT and HA-Vps27^K>R) was assessed in ubp2Δ cells expressing myc-Ub. Anti-HA (Vps27) immunoprecipitates (IP) were immunoblotted (IB) for HA-Vps27 (anti-HA) or Ub (anti-myc). Input represents a 5% equivalent. *, expected molecular weight; **, ubiquitinated forms. (D) Sorting of Ste3-GFP in vps27Δ, vps27Δ ubp2Δ, or vps27Δ hse1Δ (ESCRT-0Δ) cells transformed with plasmids expressing HA-Vps27^WT or HA-Vps27^K>R. Bar, 5 µm.

Figure 2.

Cells lacking Rsp5 activity still sort ubiquitinated cargo proteins into vacuoles. (A) Wild-type and temperature-sensitive rsp5-1 cells expressing HA-Vps27 and Myc-Ub were shifted to 37°C for 1 h. Input lysates and Vps27 immunoprecipitates (IP) were immunoblotted (IB) for HA (Vps27) and Myc (Ub). Twice the amount of the anti-HA IP from rsp5-1 cells was loaded. *, expected molecular weight; **, ubiquitinated forms. (B) Localization of Mup1-GFP and Mup1-GFP-Ub in mutant EN67 cells lacking multiple Rsp5 adaptors. Cells were grown in YPD and copper for 4 h to produce Mup1 fusion proteins from the CUP1 promoter. (C) Sorting of Gap1-GFP, Mup1-GFP, and Mup1-GFP-Ub after Rsp5 inactivation. Cells (rsp5-1) were shifted to 37°C for 1 h and grown in YPD in the presence of 100 µM CuCl₂ to induce production of GFP-tagged proteins under control of the CUP1 promoter. (D) Localization of Gap1-GFP, Ste3-GFP, Mup1-GFP, Ste3-GFP-Ub, or Mup1-GFP-Ub in rsp5Δ-null cells grown in YPD + 25 µM CuCl₂. (E) Localization of Mup1-GFP or Mup1-GFP-Ub^K63R in cells expressing wild-type (WT) Ub (SUB492) or K63R Ub (SUB493) as their sole source of Ub. Bars, 5 µm.

Figure 3.

Fusing cargo to the catalytic domain from deubiquitinating enzymes blocks ubiquitination and trafficking to the vacuole. (A) Schematic of DUb catalytic domain fusion proteins (top). Localization of the indicated GFP-tagged proteins fused to either enzymatically active or inactive (*) catalytic domains of Ubp7, HSV-1 UL36, and mCMV M48 (bottom). (B) The effect of DUb fusion proteins (Ste3-RFP-Ubp7 or Fur4-RFP-Ubp7) on coexpressed Gap1-GFP or Ste3-GFP. (C) Cycloheximide chase with cells expressing HA-tagged Ste3-GFP-m48* and Ste3-GFP-M48, which carried active and inactive DUb domains, respectively. Aliquots were removed at the indicated times after cycloheximide addition (100 µg/ml) and immunoblotted for HA and PGK. (D) Ste3-GFP-UL36 or Ste3-GFP-ul36* (with an inactive catalytic domain) were expressed in end3Δ cells and immunoblotted with anti-GFP antibodies (left) or localized (right). (E) Localization of Ste3-GFP or Ste3-GFP-Ubp7 in wild type, rcy1Δ cells, and vps4Δ cells . Bars, 5 µm.

Figure 4.

Fusion of DUbs to ESCRT proteins inhibits their ubiquitination but allows vacuolar sorting of Ub fusion cargo proteins. (A) Schematic of the ESCRT sorting apparatus showing subunits fused to different DUb catalytic domains. (B) Cells expressing myc-Ub and either Hse1-UL36-HA or Hse1-ul36*-HA (inactive) were transformed with vector alone or with plasmids expressing HA-Vps27. Lysates were immunoprecipitated (IP) with anti-HA antibodies. Immunoprecipitates were immunoblotted (IB) for HA (top) to confirm isolation of HA-tagged proteins; immunoprecipitates were immunoblotted with anti-myc to assess ubiquitination. Fusion of active UL36 abolished ubiquitination of either HA-tagged Hse1 or Vps27 proteins. Black lines indicate that dividing lanes have been spliced out. (C) ESCRT subunits, GGA, or His3 fused to the indicated active (Ubp7 or UL36) or inactive (ubp7* or ul36*) catalytic domains (CD) were expressed in wild-type cells. Cells coexpressed the indicated GFP-tagged MVB cargo. Bar, 6 µm.

Figure 5.

The effect of localizing DUb domains on ESCRTs and the Ub ligase Rsp5. (A) Complementation of mvb12Δ by Mvb12-UL36. The localization of Fur4-GFP and Ste3-GFP-Ub in mvb12Δ vps23ΔUb vps36ΔUb cells carrying vector plasmid alone or plasmids expressing Mvb12 fused to active (UL36, Ubp7) or inactive (ubp7*) DUb domains is shown. (B) Complementation of hse1Δ by Hse1-UL36 and Hse1-ul36*(inactive). The localization of Ste3-GFP and Ste3-GFP-Ub in hse1Δ cells carrying the indicated plasmids is shown. (C) Complementation of vps23Δ by Vps23-FP-UL36. Localization of Gap1-GFP and Fur4-RFP-Ub in vps23Δ cells carrying indicated plasmids is shown. (D) Wild-type cells expressing Rsp5 fused to active (Ubp7) or inactive (ubp7*) catalytic DUb domain and the indicated GFP-tagged MVB cargo proteins. Expression of Rsp5 fusions were from the CUP1 inducible promoter, induced 5 h before microscopy (for Rsp5-Ubp7) or overnight (for Rsp5-ubp7*). Bars, 5 µm.